Recent advances and challenges in single cell protein (SCP) technologies for food and feed production

The global population is increasing, with a predicted demand for 1250 million tonnes of animal-derived protein by 2050, which will be difficult to meet. Single-cell protein (SCP) offers a sustainable solution. This review covers SCP production mechanisms, microbial and substrate choices, and advancements in metabolic engineering and CRISPR-Cas. It emphasizes second-generation substrates and fermentation for a circular economy. Despite challenges like high nucleic acid content, SCP promises to solve the global nutrition problem.

typical composition of the "classic" microorganisms useful for SCP production: fungi, algae, yeast (which belong to the fungus kingdom), and bacteria 8 .First-generation SCP primarily involves the utilization of microorganisms, such as bacteria, yeast, and fungi, to convert various organic substrates, such as sugar, into biomass, comparable to first-generation biofuels.These microorganisms are cultivated under controlled conditions, optimizing their growth and protein production.On the other hand, the second generation of SCP explores innovative approaches, including nonfood feedstocks such as agricultural residues, into protein-rich biomass 9 , as well as deploying genetically modified microorganisms and novel bioreactor designs, to enhance protein yields and reduce production costs 10 .Also, the production of specialty proteins, e.g., bioactive peptides, is being investigated.By harnessing advancements in biotechnology and fermentation techniques, researchers aim to overcome limitations associated with firstgeneration SCP, such as substrate availability and process scalability.SCP is part of cellular agriculture, but different from lab-grown meat (cultured meat), where animal cells are grown in bioreactors.The focus of SCP is protein; While the inactivated cells can be fed to animals directly, SCP for food entails the further purification of the protein.
Single-cell proteins have emerged as a promising solution to address the growing demand for protein sustainably 11 , by allowing low-cost production with minimized demand for resources.They can be cultivated using a variety of substrates, including agricultural waste and industrial by-products, thus reducing pressure on traditional agricultural practices and mitigating issues related to land and water usage 12 .With ongoing advancements in biotechnology and fermentation processes, the commercial viability and scalability of SCP production are steadily improving, paving the way for their integration into mainstream food systems and contributing to global food security efforts 13 .
Single-cell proteins contain essential vitamins, amino acids, minerals, nucleic acids, and lipids, making them versatile for various applications, from food and feed production to additional applications in further industries such as the paper industry 14 .The primary sources for SCP include sugars/starch (comparable to first-generation biofuels), but also various side and waste streams, such as fruit waste, molasses, etc., as well as combustible waste and by-products like methane, methanol, and ethanol 15 .In this respect, SCP by gas fermentation is particularly interesting, as CO/H 2 (syngas) and CH 4 (biogas or obtained from CO 2 via methanation) can be obtained from a broad range of waste biomass streams, and H 2 /CO 2 allows for the recycling of carbon dioxide from (industrial) point sources or direct air capture.Utilizing waste for SCP production has numerous advantages, including converting low-cost organic materials into valuable products and reducing environmental burden 11 .In this manuscript, the term "waste" is used in a broad sense to encompass various types of biomass residuals.For "co-products" or "by-products" being permissible for use in food and feed production, the relevant legislation needs to be born in mind.In the EU, for instance, the key regulatory frameworks governing these aspects include the General Food Law Regulation (EC) No 178/2002, which establishes the principles of food and feed safety, and the Feed Hygiene Regulation (EC) No 183/2005, which sets the requirements for feed production.For example, Regulation (EU) No 68/2013 on the Catalog of Feed Materials specifies the conditions under which various materials, including co-products and byproducts, can be used in feed.These materials must be safe, of good quality, and properly labeled.Therefore, while co-products and by-products can be used in food and feed production, they must comply with the relevant safety, quality, and traceability regulations.
Agricultural waste and side streams, such as waste wood with its components cellulose, hemicellulose, and lignin, serve as excellent natural sources of SCP but require pretreatment through chemical (acid hydrolysis) or enzymatic (cellulases) methods to transform the raw materials into fermentable sugars 16 .Additionally, domestic sewage and waste from industrial processes, e.g., starch production and food processing, can be repurposed for SCP production 17 .Agricultural waste not only provides a cost-effective substrate for SCP production, producing protein-enriched materials suitable for animal feed but with further processing, it can also be consumed by humans 18 .While first-generation substrates focus on enhancing protein security in the face of a growing global population, from edible substrates, second-generation substrates contribute to the evolving circular economy and sustainability of the food system 11 .These substrates involve the renewable bioconversion of waste or underutilized co-products into highvalue microbial protein products 19 .Various fermentation technologies, including solid, liquid, and semisolid methods, have been explored for cultivating diverse strains of yeast, bacteria, algae, and filamentous fungi, with process outcomes influenced by fermentation conditions, substrate characteristics, and microbial types 20 .
The consumption of SCP, especially those materials derived from algae, has garnered increasing interest due to their potential as a sustainable protein source with numerous nutritional benefits 7 .Table 2 highlights global algae consumption as a historical food source in various countries.Both macro-and microalgae have been on humans' diets.Macroalgae contain varying levels of nutrients depending on species, season of harvest, geographic origin, and environmental conditions 21 .The protein and nutritionally essential amino acids content can be rather low and variable, especially in brown (Phaeophyta), green (Chlorophyta), and red (Rhodophyta) algae macroalgae, when considered against the amino acid requirement of most aquacultural and terrestrial animal species.It is reported that marine macroalgae are rich in protein content 22 .Recent research findings have revived interest in marine macroalgal biomass as a potentially sustainable feedstock for protein production.Comparing the protein content of macroalgae reported in different studies can be difficult owing to methodological differences 23 .However, the protein content of brown macroalgae is generally low (usually under 150 g kg −1 of dry matter (DM)), whereas green macroalgae, and especially red macroalgae, have a higher protein content on a DM basis.Some red macroalgae, such as Porphyra spp., contain protein levels comparable to soybean meal 18 , which is around 50% on a DM basis.
Nielsen et al. 24 reported that species of green algae sea lettuces (Ulva genus) have been the subject of several animal experiments.Intact biomass from Ulva spp. is relatively rich in protein and has potential as an alternative protein source in animal feed.Seaweed-derived protein may offer a viable solution as seaweeds may be considered sustainable and highly nutritious, and they do not require land for cultivation 25 .Seaweeds have a reported protein content of between 8 and 47% dry weight, and the highest protein content is found in red and green seaweed species 26 .Red macroalgae of the genus Porphyra, e.g., P. purpurea, P. yezoensis, and P. dioca, have been studied as protein sources in diets.Previous research 27 included P. purpurea meal containing 250 g kg −1 of protein at 165 and 330 g kg −1 in isonitrogenous and isoenergetic diets (i.e.same nitrogen and energy contents) for the omnivorous thick-lipped gray mullet (Chelon labrosus), replacing fishmeal.The results showed that body weight gain, specific growth rate, feed efficiency, and protein efficiency ratio as well as net protein utilization decreased with increasing P. purpurea inclusion levels.The protein content of macroalgae varies within seasonal cycles.One example is the protein content of the red seaweed Palmaria palmata collected on the French Atlantic coast, which showed fluctuations between 9 and 25% dry weight in protein content.The highest values occurred during winter and spring 28 .Macroalgae, rich in diverse nutrients and proteins, have gained attention as a potential sustainable alternative to conventional protein sources.Microalgal biomass production has been long considered a promising way to close the predicted 'protein gap'.Reported crude protein content of microalgal biomass varies between 30 and 80 mass percent: for example, Chlorella vulgaris, 51-58%; Arthrospira platensis 60-71%; Tetraselmischui 31-46%; Nannochloropsis oceanica 35-44%; Dunaliella salina, 50-80%; Galdieria sulphuraria, 62% 29 .As such, microalgal protein content generally is higher than that of dried skimmed milk (36%), soy flour (37%), chicken (24%), fish (24%), and peanuts (26%) 30 .Despite the high-protein content and the favorable amino acid composition, direct use of microalgal biomass as single-cell protein (SCP) is limited by its digestibility 31 .Galdieria sulphuraria is an extremophile red microalga that thrives in acidic environments with pH values from 0 to 4 and temperatures up to 56 °C32 .It is reported that G. sulphuraria has great potential for the biotechnological production of pigmented proteins like phycocyanin 33 .There are proteins synthesized by G. sulphuraria of biotechnological interest, including phycobiliproteins (phycoerythrin, allophycocyanin, and phycocyanin).
However, practical barriers, such as the great difficulty of extracting proteins from microalgae, exist.The separation of microalgae proteins from cellular debris is mainly based on their dispersibility in water and the separation of the protein-rich aqueous phase from the solid phase 34 .Microalgae have complex cell wall structures that can be difficult to break down, making it challenging to access and extract proteins efficiently 35 .Traditional protein extraction methods can be expensive and energyintensive, reducing the economic feasibility of large-scale protein production from microalgae 36 .It is reported that various extraction methods, including mechanical disruption, enzymatic digestion, and chemical treatments, are used for protein extraction from microalgae 37 .Each method has its advantages and drawbacks.Mechanical disruption, such as bead milling or ultrasonication, can break down the cell walls but may also damage the proteins and require additional purification steps.Enzymatic digestion can selectively target cell wall components but may be slow and costly.Chemical treatments may be efficient but raise concerns about residue removal and environmental impact 38 .
There was considerable interest in algae for biomass and biofuel production a long time ago, however much of the euphoria has subsided.There are several reasons why the initial enthusiasm for algae as a source of biomass and biofuel may have faded, such as technical challenges, economic viability, environmental concerns, and market dynamics 39 .
Cyanobacteria are also utilized for the production of single-cell proteins.These single cells are characterized by a low content of nucleic acids and a high amount of proteins.A cyanobacterium grows comparatively rapidly and produces good-quality proteins.It has been demonstrated that Spirulina maxima and Spirulina platensis are the most important cyanobacteria, which contain 60-70% protein content 40 .Due to the high quality and quantity of proteins obtained from Spirulina, it is very useful to human nutrition.Also lend themselves to the production of single-cell oil (SCO).
Table 3 outlines various substrates utilized for cultivating different organisms, as common practice in the production of single-cell proteins 41 .The choice of protein derived from diverse species is grounded in its chemical composition (e.g. protein and fat content) and its amino acid profile 42 .Fungi, particularly when cultivated for single-cell protein production, contain a substantial 30 to 50% protein content 43 .Their amino acid profiles adhere to the standards set by the FAO, generally characterized by richness in lysine and threonine but a deficiency in cysteine and methionine, which are sulfur-containing amino acids primarily derived from plant sources 44 .Noteworthy is the fungus K. fragilis, which demonstrates the ability to produce sulfur-containing amino acids when grown on whey 45 .Single-cell protein from fungi not only provides protein but also additional nutrients, including various B-complex vitamins such as riboflavin, niacin, thiamine, biotin, pantothenic acid, choline, pyridoxine, glutathione, pamino benzoic acid, streptogenin, and folic acid 46 .However, it is important to note that feeding on mycoprotein obtained from Fusarium venenatum has been reported to lead to a decrease in insulin and blood glucose levels 47 .Fungi also tend to have a relatively higher nucleic acid content, ranging from 7 to 10%, compared to algae 48 .Fusarium venenatum is used to produce the mycoprotein Quorn™, which was launched in 1985 by Marlow Foods.
Bacteria, with an exceptionally short generation time of 20 to 120 min, exhibit rapid multiplication and can grow on a diverse range of raw Table 3 | List of substrates for various bacteria, algae, and fungal species [30][31][32][33][34][35][36][37][38][39] materials and edible (and also non-edible) substrates such as starches and sugars 49 .Bacteria thrive on organic matter, petrochemical wastes (hydrocarbons), and other cheap substrates, including ethanol and methanol, as well as methane and carbon monoxide.Certain bacteria, like Methylophilus spp., exhibit an impressively short generation time of 2 h and are valuable components of animal feed, offering protein that is chemically superior to yeast or fungi 50 .
This paper extensively reviews the current landscape of single-cell protein (SCP) technology, grouped into eight main sections.The initial sections (first to third) focus on the production perspective, addressing production mechanisms, key steps, and the involvement of relevant microbes and substrates, with a focus on recent advancements in the field.The subsequent sections (fourth and fifth) shift the focus to genetic studies related to SCP production and an examination of its sustainability prospects.The sixth section provides an update on commercial activities in the realm of SCP, while the seventh section explores feed and food legislation.The final sections (eighth and ninth) delve into the progress of sustainability research, comparing results among different countries, and addressing SCP challenges.The article concludes by summarizing key findings and highlighting future research priorities.
A comprehensive literature search was undertaken utilizing multiple academic databases, including Web of Knowledge, Scopus, ScienceDirect, Google Scholar, Web of Science, and ResearchGate.The search spanned from 2016 to early 2024, focusing on various keywords related to single-cell protein, fermentation, microalgae, microbial protein, yeast, bacteria, solidstate fermentation, biomass production, wastes, and substrates.To refine the search and obtain precise results, operators like "OR" and "AND" were employed, and specific terms, such as "methane fermentation" or "gas fermentation," were enclosed in quotation marks.References cited within the acquired publications were also collected to ensure a thorough literature review.The chosen keywords were consistently applied across all four databases to maximize search coverage and compile and analyze a comprehensive collection of literature on SCP.Bibliometrix™ was employed to identify trending keywords and topics in each specific review section.A total of 130 articles were screened and evaluated based on titles and abstracts, employing a stringent selection process that prioritized studies with quantitative information.The targeted studies addressed single-cell protein production mechanisms and sustainable production methods, challenges, and future perspectives.Following a thorough comparison and analysis of the remaining articles, they were categorized based on relevant keywords.The conducted research was summarized, and the content of the 130 studies was critically evaluated.Essential features were extracted, and key challenges warranting further investigations were identified.Through this rigorous methodology, the aim was to offer a comprehensive review that synthesizes the current state of knowledge, emphasizes significant findings, and pinpoints areas necessitating additional research attention within the context of single-cell protein mechanisms.Figure 1 summarizes the bibliometric analysis of the review sections and Fig. 2 shows the preferred reporting items for systematic reviews and meta-analyses (PRISMA) flow diagram of this study.
The mechanism involved in the production of single-cell protein (SCP) Figure 3 offers a simplified visual representation illustrating the sequential process flow involved in SCP production.The production journey of SCP begins with substrate preparation, a pivotal step in converting substrates into a usable carbon source 51 .The approach to substrate preparation is contingent upon the type of substrate and the selected fermentation method 52 .Recent literature introduces second-generation substrates (SGS) prepared through diverse methods, including wet, direct, and dry preparation 53 .These methods are intricately linked with liquid-state fermentation, solid-state fermentation, and semisolid-state fermentation, respectively 54 .The wet preparation method is commonly applied to substrates with high moisture content, such as fresh fruit or vegetable waste 55 .It involves a sequence of water or acidic washes, pulverization, filtration, and sterilization to yield a sterile liquid medium 56 .For low-moisture substrates, the dry method is employed, utilizing a drying procedure to eliminate moisture.This is followed by a reduction and sifting process, producing a fine powder with defined particle sizes 57 .The fine powder is then combined with water, filtered, and sterilized, resulting in a sterile moist-solid medium with a significant aqueous phase 58 .
The direct method is chosen when adopting the solid-state fermentation (SSF) approach 59 .It encompasses water washing and hydrolytic processes, including acid, bio hydrolysis, or thermal treatment, to convert the substrate into a fermentation-ready medium 60 .Lignocellulosic materials, due to their low-moisture content and bulkiness, often undergo dry and direct preparation methods 61 .However, the grinding and digestion of the substrate remain vital in their preparation to enhance nutrient accessibility, considering the complex interaction in their chemical composition 62 .Sterilization of the resultant media is conventionally carried out using an autoclave at a temperature of 121 °C for ~15-60 min, with the autoclaving duration adapted based on the substrate and contamination levels 63 .
After the substrate preparation stage comes media enrichment, a process designed to enhance the nutritional content of the resulting medium 64 .When utilizing second-generation substrates for SCP production, there has been a reliance solely on the substrate as the nutrient source for the fermentation process, leading to optimal microbial doubling without additional enrichment 65 .This suggests the potential option of bypassing the media enrichment step 66 .The subsequent stage is the inoculation phase, which may involve isolating or cultivating microbial cells to attain a substantial load before introducing them to the prepared medium 67 .Alternatively, a viable inoculum can be acquired from suppliers and used directly, or the back-slopping procedure can be employed as necessary 68 .Following inoculation, the medium undergoes incubation (fermentation).Throughout this incubation period, the fermentation environment is intermittently regulated to optimize the multiplication of microbes 69 .The fermentation can be done with pure or mixed cultures.

Fermentation approaches
Microorganisms efficiently utilize available wastes as a growth medium to augment their cell masses, constituting the SCP 70 .The main process responsible for SCP production is fermentation, which can take the form of either submerged or solid-state fermentation 71 .Upon the completion of the fermentation process, the resulting biomass is harvested, providing a valuable protein source 72 .Subsequently, this biomass undergoes further processing techniques such as purification, cell disruption, washing, and protein extraction 73 .These steps contribute to achieving, in general, high rates of production, improved yields, and better control over the production process, e.g., nucleic acid reduction.

Solid-state fermentation (SSF)
This kind of fermentation entails the cultivation of microbes on a solid substrate without a free aqueous phase, employing various fermenter designs 74 .Microbes flourishing in SSF can thrive with minimal or no extra water, as the inherent moisture in the wet solid substrate provides sufficient conditions for their growth and metabolic activities.This characteristic makes SSF an efficient and resource-conserving process for microbial cultivation.This confines SSF to microorganisms well-suited for growth under low water activity, such as yeast 75 , and certain filamentous fungi that prefer pure solid substrates with ~60-65% moisture content 76 .
In SSF, microbes acquire nutrients through adsorption or penetration of the solid substrate, enabling them to multiply into protein-rich biomass 77 .A nutrient concentration gradient exists in SSF, necessitating nutrient diffusion for optimal access during microbial proliferation 78 .The fermentation process in SSF requires sufficient oxygen supply in the liquid phase, achieved through aeration and intermittent medium stirring, alongside maintaining optimal conditions for temperature, pH, ionic strength, and nutrients to ensure maximum yield 79 .This kind of fermentation offers advantages due to its relatively low capital investment and minimal waste generation, delivering economic and environmental benefits 80 .Consequently, https://doi.org/10.1038/s41538-024-00299-2 Review article npj Science of Food | (2024) 8:66 contemporary fermentation-based research is increasingly favoring the SSF approach to enhance techno-economic and sustainability benefits 81 .However, SSF encounters substantial challenges requiring significant innovations to unlock its full commercial potential 82 .Key challenges include difficulties in scale-up, currently hindering large-scale production, and limitations in online monitoring and process control 14 .Moreover, addressing issues related to stirring and removing metabolic heat in SSF has been crucial for improving process efficiency, and recent developments hold promise for better system designs to overcome these limitations 83 .Figure 4 provides a schematic illustration of the processes that take place during SSF with fungi.
Solid-state fermentation indeed offers several advantages, including potentially higher product yields, lower water usage, and reduced energy requirements compared to traditional submerged fermentation methods.While solid-state fermentation shows promise as a sustainable and efficient bioprocessing method, its successful implementation at a large scale requires careful evaluation of technical, economic, and market factors.It is essential to acknowledge the challenges associated with scaling up such processes to industrial levels.Furthermore, it is crucial to conduct comprehensive techno-economic analyses (TEAs) to assess the production costs and competitiveness of products derived from solid-state fermentation compared to existing alternatives.

Liquid-state (submerged) fermentation (LSF)
Unlike SS, LSF, also referred to as submerged fermentation, involves microbial cultivation in a continuous liquid-phase substrate with more than 95% water content 84 .Conducted in a closed bioreactor, typically in continuous mode, this submerged process ensures precise control over temperature, pH, nutrients, and oxygen supply 85 .It is widely adopted in industrial fermentation processes due to its advantages in comparatively easy scale-up, uniform distribution of nutrients and oxygen facilitated by the continuous liquid phase, and the ability to achieve high-protein yields 86 .Furthermore, submerged fermentation allows for the efficient removal of metabolic heat and online monitoring or control of the process 87 .
However, despite these advantages, the characteristic high capital demand and significant waste generation associated with submerged fermentation are gradually diminishing its appeal in an evolving sustainability-sensitive economy 88 .Although research and technological advancements are addressing these limitations to enhance system performance, the increasing emphasis on the SSF approach, driven by its sustainability prospects, raises the possibility that SSF may outpace LSF in future industrial adoption 89 .Yet, this potential transition may face challenges 90 .

Semisolid-state fermentation
Semisolid-state fermentation represents an intermediary approach between SSF and LSF 91 .In this method, the free-flowing liquid content is increased to enhance the distribution of nutrients and oxygen 92 .This positioning makes it a preferred intermediate method for microbes that require a slightly higher water activity but perform better on a solid substrate 93 .However, as an intermediate approach, its advantages and disadvantages fall between SSF and LSF 94 .For instance, while it offers moderate metabolic heat removal facilitated by its slightly higher liquid phase relative to SSF, it is characterized by a high capital investment compared to SSF and a lower protein yield compared to LSF (Table 4).Traditional fermentation.Traditional fermentation is a form of indigenous and spontaneous fermentation that involves the natural growth of microorganisms and their metabolites 95 .Lactic acid bacteria and yeasts are the major microorganisms involved in these spontaneous fermentations 96 .However, in the traditional condiments from alkaline fermentation, investigations have shown that microorganisms such as Bacillus sp. and Staphylococcus sp. were predominant 97 .Globally, during fermentation, microorganisms act on the available carbohydrates, converting them into organic acids, alcohols, carbon dioxide as well as bacteriocins 98 .Since traditional fermented foods regained great interest in the general public and scientific world, many other functional properties of these foods and the microorganisms involved in their fermentation have been revealed.The fermentation process is known to increase flavor, nutritional quality, preservative effects, detoxication, and medicinal properties 99 .Traditional fermentation improves the digestibility of raw products, eliminates useless compounds, and increases the bioavailability of proteins, lipids, carbohydrates, minerals, and vitamins as well 100 .It is reported that traditional fermented foods and beverages provide a significant source of nutrients depending on the substrate used for their production 101 .Fermented foods based on soybean and cabbage (chungkookjang, doenjang, meju, gochujang, natto, sauerkraut, tempeh) are a source of protein, essential fatty acids (linoleic and linolenic), soluble fiber, minerals such as iron and zinc, as well as vitamin K, vitamin B9, B1, and B6.Similarly, certain fermented foods made from milk (yogurt, kefir, dahi) contain mainly high-biological value proteins, calcium, and vitamins B2, B12, and B9 102 .In contrast, fermented foods made from cereals (pozol, red yeast rice) possess essential amino acids (threonine, valine, isoleucine, and leucine) and insoluble fiber, as shown in Fig. 5. Furthermore, traditional fermented foods play a crucial role in promoting gut health by serving as probiotics, enhancing the gut microbiota's diversity and balance 103 .This is particularly significant in today's context, where gut health is increasingly recognized as central to overall well-being, with implications for immune function, mental health, and more 104 .Moreover, the fermentation process can lead to the production of bioactive compounds with potential health benefits, such as antimicrobial peptides, antioxidant compounds, and anti-inflammatory agents 105 .These bioactive compounds contribute to the functional properties of fermented foods, making them not only nutritious but also potentially therapeutic 106 .As consumers become more health-conscious and seek natural, minimally processed foods, the demand for traditional fermented products is likely to continue to grow, driven by both their cultural significance and their perceived health benefits 107 .

Biomass fermentation in alternative protein production
Biomass fermentation is pivotal in the alternative protein sector, particularly for the sustainable production of high-protein, fibrous microbial biomass 108 .This process leverages the rapid growth capabilities of specific microorganisms, such as filamentous fungi, yeast, and bacteria, to produce substantial quantities of protein-rich biomass 109 .These microorganisms are cultivated in controlled fermentation processes where their growth is maximized to yield biomass that is inherently rich in proteins and can be directly utilized as food ingredients 110 .
One of the primary applications of this technology is the production of "one-cut" meat substitutes, where the cultivated fungal biomass, due to its fibrous nature, serves as an excellent alternative to traditional meat 111 .The texture and nutritional profile of the produced biomass make it suitable for integration into various food products, offering a sustainable and environmentally friendly protein source that reduces reliance on conventional meat and dairy products 112 .
Furthermore, the versatility of biomass fermentation extends to the production of other valuable bioproducts such as enzymes and organic acids 113 .However, the focus in the context of alternative proteins remains on enhancing the scalability and efficiency of producing microbial biomass that meets the nutritional demands and culinary expectations of consumers worldwide 114 .This transformative approach not only aligns with sustainable development goals but also paves the way for a bio-based economy, where renewable biological resources are utilized efficiently to address nutritional needs and minimize environmental impacts 115 .
The various stages of this biomass fermentation process, including pretreatment, enzyme production, and the maximization of microbial growth, are detailed in Fig. 6.This figure illustrates the integration of bioprocessing steps, emphasizing the controlled conditions under which microbial biomass is cultivated for food applications.

Gas fermentation
Gas fermentation is a promising bioprocess that involves the conversion of gaseous substrates, such as carbon monoxide (CO), carbon dioxide (CO 2 ),

Dry method
Wet method Direct method Fig. 3 | A generic, simplified production flow chart of SCP that can be used directly for feed but requires additional downstream processing for food.This flow chart provides a simplified overview of the production process of single-cell protein (SCP). https://doi.org/10.1038/s41538-024-00299-2 Review article npj Science of Food | (2024) 8:66 or hydrogen (H 2 ), into biomass products through microbial activity 116 .This innovative approach has gained traction as a means of utilizing waste gases from industrial processes or renewable resources for the production of biochemicals and other high-value compounds 117 .Gas fermentation represents a sustainable alternative to traditional fermentation processes reliant on agricultural feedstocks and offers opportunities to reduce greenhouse gas emissions and mitigate environmental impacts 118 .At the heart of gas fermentation lies a diverse array of microorganisms capable of metabolizing gaseous substrates and synthesizing target products 119 .One of the most extensively studied pathways in gas fermentation is the Wood-Ljungdahl pathway, also known as the reductive acetyl-CoA pathway, which enables the conversion of carbon monoxide or carbon dioxide into acetyl-CoA, a central metabolic intermediate 120 .Microorganisms equipped with this pathway, such as acetogens and homoacetogens, can produce a range of compounds, including ethanol, acetate, and butanol, from carbon monoxide or carbon dioxide as the sole carbon source 121 .Hydrogenotrophic microorganisms, such as certain archaea and bacteria, specialize in utilizing molecular hydrogen as an energy source for growth and metabolism 122 .These microorganisms play vital roles in biogas fermentation processes, where hydrogen is generated through the anaerobic digestion of organic matter 123 .Methanogenic archaea further metabolize hydrogen and carbon dioxide to produce methane, the primary component of biogas 124 .Gas fermentation systems harness these microbial consortia to convert waste gases into methane-rich biogas for energy production 125 .
In addition to biomass production, gas fermentation holds promise for the synthesis of platform chemicals and specialty compounds with diverse industrial applications 126 .Microorganisms engineered to express specific enzymes or metabolic pathways can produce a wide range of products,  including organic acids, alcohols, and hydrocarbons, from gaseous substrates 127 .These bioproducts serve as renewable alternatives to petrochemical-derived counterparts and contribute to the transition toward a bio-based economy 128 .Despite its potential, gas fermentation faces challenges related to process optimization, microbial strain development, and product recovery 129 .Efforts are underway to enhance the efficiency and scalability of gas fermentation technologies through advancements in microbial engineering, bioreactor design, and downstream processing techniques 130 .As research progresses and technological barriers are overcome, gas fermentation is poised to emerge as a key strategy for valorizing waste gases and enabling the sustainable production of fuels and chemicals 131 .Bacteria fermenting CO 2 -and CO-rich gases have a range of natural products that are mainly of interest as biocommodities or biofuels, and some are already on their way to being commercialized 132 .Acetate is the typical product of acetogenic bacteria and advances have been made to improve the production of natural acetate producers by genetic modification 133 .The Wood-Ljungdahl pathway is shown in Fig. 7.

Genetics studies in single-cell protein
Metabolic engineering is pivotal in optimizing the biotechnological production of SCP from bacteria, fungi, and algae.This strategy involves deliberate modifications to microbial metabolic pathways to enhance protein synthesis.Specifically, researchers focus on genes related to amino acid biosynthesis, protein folding, and nitrogen assimilation 134 .The optimization of amino acid biosynthesis aims to bolster the cellular pool of essential amino acids crucial for robust protein construction 134 .Genetic interventions such as overexpressing key biosynthetic pathway genes ensure a surplus of specific amino acids, contributing to efficient protein synthesis 135 .Meanwhile, ensuring proper protein folding is paramount for functional and stable proteins.Metabolic engineering addresses this by targeting genes involved in protein folding mechanisms, potentially adjusting chaperone protein expression or other folding factors 136 .This optimization mitigates the formation of misfolded or aggregated proteins, enhancing overall protein quality 137 .
Moreover, nitrogen assimilation plays a fundamental role in amino acid and protein synthesis.Metabolic engineering intervenes in bacterial nitrogen assimilation pathways to optimize nitrogen utilization for protein production 138 .By manipulating genes related to nitrogen uptake and assimilation, researchers ensure readily available nitrogen, crucial for amino acid and subsequent protein formation 139 .Techniques such as recombinant DNA technology and CRISPR-Cas9 enable precise genome editing, facilitating targeted modifications in protein synthesis-related genes 140 .Additionally, synthetic biology approaches aid in designing novel metabolic pathways for optimal protein production 141 .
The metabolic engineering's benefits in SCP production are multifaceted.Increased protein yield, achieved by enhancing specific pathways, and resource-efficient production processes are prominent outcomes 142 .Customizing microbial metabolism tailors growth conditions and protein content to meet specific SCP production requirements 143 .The benefits of metabolic engineering in SCP production are multifaceted.Increased protein yield is a prominent outcome, achieved by enhancing specific pathways crucial for protein synthesis 144 .The optimization of metabolic pathways ensures efficient nutrient utilization, making the production process more resource-effective 145 .Metabolic engineering allows the tailoring of the microorganisms' metabolism to meet the specific requirements for SCP production, optimizing growth conditions and protein content 146 .Feed and food approval with genetically modified organisms (GMO) is more complex than with naturally occurring strains.
The approaches described here can indeed be applied to various sources of SCP, including bacteria, fungi, and algae, which are representative of both first and second-generation SCP sources 147 .Metabolic engineering techniques are versatile and can be adapted to different microbial species to enhance protein synthesis and optimize production processes 148 .Regarding the potential applications for food and feed uses, the versatility of metabolic engineering allows for customization based on the intended purpose 149 , also known as precision fermentation Whether the SCP is destined for human consumption or animal feed, the optimization of metabolic pathways can ensure the production of high-quality protein with efficient nutrient utilization 150 .Therefore, these approaches can be tailored to meet the specific requirements of both food and feed applications, contributing to sustainable protein production across various sectors.
Exploring the RNA targeting process using the singleprotein CRISPR effector Cas7-11 RNA interference (RNAi) is a fundamental cellular process primarily observed in eukaryotic organisms, responsible for the selective regulation of gene expression by effectively silencing specific genes.This intricate mechanism plays a crucial role in maintaining cellular functions and responding to various biological processes in eukaryotes.In the context of SCP production, while RNAi may not be directly applicable in prokaryotes, similar mechanisms of gene regulation exist, such as CRISPR-Cas systems.
These systems can be employed to downregulate genes associated with undesirable traits or to fine-tune the expression of genes involved in protein synthesis in prokaryotic hosts 151 .This precision in gene regulation contributes to the optimization of microbial strains for enhanced SCP production.For instance, in prokaryotic hosts, CRISPR-Cas systems can be utilized to influence the composition of SCP by selectively enhancing the expression of genes responsible for desirable nutritional attributes.By targeting specific genes involved in amino acid synthesis or bioactive compound production, researchers can tailor SCP to meet specific nutritional requirements for various applications 152 .Additionally, CRISPR-Cas systems offer a powerful tool for mitigating stress responses encountered by microbes during SCP production.By modulating the expression of stress-related genes, microbial cells can maintain optimal protein production levels even under challenging growth conditions, ensuring consistent SCP yields 153 .
CRISPR-Cas and RNAi technologies play pivotal roles in advancing SCP production, albeit in different cellular contexts.They empower researchers to engineer microbial strains with precision, optimizing metabolic pathways and gene expression to meet the evolving demands for sustainable and high-quality protein sources 154 .The application of these technologies ensures that SCP remains a promising solution for addressing global protein challenges 155 .
The CRISPR/Cas systems, primarily prokaryotic and categorized into two classes and six major types, spotlight the class 2 type II CRISPR system (CRISPR/Cas9) 156 .This specific system is extensively researched and developed as a versatile toolbox for gene editing and other applications, as indicated by Fig. 8.As a eukaryotic model microorganism, S. cerevisiae was one of the earliest hosts for CRISPR/Cas9-mediated genome editing 156 .To improve genome editing efficiency in eukaryotic hosts, considerations such as codon usage and nuclear localization sequences become crucial (Fig. 8).
The utilization of CRISPR-Cas systems in prokaryotic hosts for SCP production offers several advantages, including the ability to precisely target and modify specific genetic elements within microbial genomes.This targeted approach allows for the engineering of microbial strains with enhanced protein synthesis capabilities and improved nutritional profiles, ultimately leading to the production of SCP with higher yields and nutritional quality 157,158 .Moreover, CRISPR-Cas systems enable the manipulation of metabolic pathways involved in SCP production, facilitating the optimization of cellular processes to maximize protein output while minimizing resource utilization and waste production.
In contrast, the application of RNAi in eukaryotic organisms provides researchers with a powerful tool for fine-tuning gene expression and regulating cellular processes relevant to SCP production.By selectively silencing genes associated with undesirable traits or stress responses, RNAi allows for the development of microbial strains with improved protein synthesis capabilities and increased resilience to environmental challenges 159 .Furthermore, RNAi-mediated gene regulation can be used to enhance the nutritional quality of SCP by promoting the synthesis of specific amino acids or bioactive compounds essential for human nutrition.The integration of CRISPR-Cas and RNAi technologies into SCP production represents a significant advancement in biotechnology.These tools offer researchers unprecedented control over microbial genomes, enabling the development of tailored microbial strains optimized for SCP production.By harnessing the power of gene editing and gene regulation, scientists can address key challenges in protein production and contribute to the sustainable and efficient production of high-quality protein sources for various applications.
Microalgae metabolism encompasses biochemical mechanisms and nutrient transport, converting intake nutrients into vital components for growth, reproduction, and defense 160 .Distinguished by oxygen-evolving photosynthesis, microalgae exhibit specific reactions at the thylakoid membrane, occurring in the presence of light.The chemo-organotrophic metabolism in microalgae resembles bacteria, with anabolic processes possible in both light and darkness 161 .Photorespiration, catalyzed by specific enzymatic systems, plays a pivotal role.The synthesis of essential carbon skeletons involves sequential pathways that utilize various components, including photons, nitrogen, and compounds like ammonium nitrate, ammonium sulfate, ammonium dihydrogen phosphate, and carbon dioxide 162 .In the absence of light, microalgae rely on organic carbon for growth.Key metabolic pathways governing this process encompass photosynthesis, photorespiration, dark respiration, and overall algal growth.Within these classes, diverse families like Spirulina, Ulva, Fucus, and Porphyra are represented, as illustrated in Fig. 9.

Environmental and social sustainability of SCP production
The preceding sections have convincingly positioned SCP as a rapidly advancing biotechnological solution for converting biomass, providing insights into its production status and promising commercial viability due to the increasing demand for protein 163 .This section further explores the sustainability aspect of SCP production, specifically examining the application of sustainability metrics and insights for continuous enhancement 164 .A thorough bibliometric exploration across diverse databases reveals the prevalent use of life cycle assessment (LCA) and techno-economic assessment (TEA) in evaluating SCP sustainability, highlighting the relatively unexplored nature of other sustainability metrics like life cycle cost assessment (LCCA), social life cycle assessment (SLCA), and environmental nutrition 165 .
Structured into three segments, the initial subsection succinctly summarizes available LCA studies, delineating their methodologies, principal findings, and relevant recommendations, while also considering the microorganism-specific impacts on environmental sustainability 166 .For instance, algae-based SCP production may offer advantages in terms of water and land use efficiency compared to traditional protein sources, whereas fungal SCP production could have lower energy requirements and greenhouse gas emissions due to its efficient growth on various substrates.Furthermore, it investigates the sustainability potential introduced by an innovative power-to-food (PtF), aka power-to-protein (PtP), technology in SCP production 167 .The second part delves into pertinent TEA studies, with a focus on economic aspects, while considering the microorganism-specific economic feasibility and scalability.For example, bacteria like Methylococcus capsulatus have been explored for their ability to utilize methane as a substrate, potentially offering cost-effective and sustainable SCP https://doi.org/10.1038/s41538-024-00299-2production methods in regions with abundant methane resources 168 .The third part identifies existing research gaps and puts forth recommendations for future studies, aiming to further deepen the comprehension and sustainable development of SCP production, taking into account the unique characteristics and potential of different microorganisms/sources.   8 | Guidelines for expressing case proteins and sgRNA in the CRISPR/Cas system.Guidelines for expressing Cas proteins and sgRNA within the CRISPR/Cas system.In the CRISPR/Cas9 system (A), the Cas9-sgRNA complex initiates a double-strand break (DSB) close to a protospacer adjacent motif (PAM), typically "NGG" for Cas9 from S. pyogenes.To efficiently target the nucleus in eukaryotes (B), it is recommended to fuse Cas9 with a nuclear localization sequence (NLS).The CRISPR/Cas12a (Cpf1) system (C) induces a DSB akin to Cas9 but relies on a different PAM ("NTTT") and generates a sticky end.For sgRNA expression (D), the use of an RNA polymerase III (RNAP III) promoter and a well-designed 20 bp spacer is essential (E), Achieving multi-sgRNA expression can be done through either multiple cassettes I or a crRNA array and tracrRNA (F), GRNA multiplexing strategies (G) incorporate various elements like RNAP II and RNAP III promoters, endonucleases, ribozymes, and a polycistronic tRNA-gRNA architecture 23 .

Life cycle assessment
https://doi.org/10.1038/s41538-024-00299-2an eco-friendly substitute for animal-based proteins, especially within the dairy and beef sectors 169 .Nevertheless, the extent of these benefits hinges on factors like substitution levels, electricity usage, and the chosen assessment boundary conditions.Renewable energy emerges as a pivotal element in maximizing SCP's environmental benefits, with recent studies indicating considerable offsets in global warming and land use 5 .Recommendations include embracing precision fermentation, leveraging virtual support systems, integrating multicriteria decision analysis, incorporating genetic engineering for microbial improvement, and expanding performance analysis to encompass nutrition, economic, and social dimensions 170 .
The study also probes into the application of innovative power-to-X (PtX) technology, specifically power-to-food (PtF) in SCP production.PtF-SCP, positioned as a self-sustaining alternative reliant on renewable energy, demonstrates a substantial 60% reduction in global warming compared to soybean production 171 .It holds promise in addressing the climate emergency and mitigating human health impacts, with substantial savings in land use, water use, and eutrophication compared to soybean production.The study underscores the significance of renewable electricity sources, such as wind and solar, in steering the environmental benefits of the PtF-SCP system.In summary, the analysis accentuates SCP's considerable environmental benefits, especially in terms of land and global warming savings 172 .Energy modeling emerges as a critical element for advancing SCP sustainability, with a preference for renewable energy and waste heat recovery systems over electricity grids heavily reliant on fossil fuels 173 .The study recognizes SCP's limitations in meeting absolute planetary boundaries, stressing the necessity for hotspot analysis and broadening impact categories to grasp trade-offs and necessary improvements for forthcoming SCP technological scenarios.

Techno-economic assessment
A recent study 174 found that SCP from stranded methane can be produced below 1600 USD/ton.Previous research 175 conducted a study on the production of SCP for food from straw, determining a minimum economically viable protein selling price between 5160-9007 €/ton dry mass of protein.Economies of scale can significantly reduce production costs, particularly those driven by fermentation and downstream processing.While specific studies on the TEA of SCP production remain limited, the integration of SCP production into existing industrial processes has shown promising economic advantages.For instance, in the context of bioethanol production, conventional methods involve converting whole stillage, a by-product, into distiller's dried grains with solubles (DDGS) energy-intensive methods.However, alternative approaches have been proposed, such as one studied by previous research 176 , which integrates SCP production into the existing bioethanol production process.In this integrated approach, a portion of the  • Across all impact categories assessed, electricity consumption emerged as the predominant contributing factor.• Undertake a consequential LCA to evaluate the production of SCP and anticipate the changes expected upon its commercialization.
• Enhance the analysis by expanding the scope of impact categories to include additional factors, such as biodiversity, closely linked to conventional protein production.
This holistic approach provides a more thorough understanding of the environmental consequences associated with the commercialization of SCP.
133 Consequential life cycle assessment to compare biorefinery pathways using organic fractions from municipal and supermarket waste.Focuses on the sustainability of valorization pathways using methane-oxidizing microbes.
Supervised and processed 1 tonne of bio-pulp, characterized by an average Total Solids (TS) content of 18.3%.This involves the management and treatment of the specified quantity of bio-pulp material.
Gate-to-gate Laboratory-scale experiments were performed, leveraging data from the Ecoinvent database (version 3.3) as the primary information source.The impact assessment method employed in the analysis was Impact 2002+, and the entire process was facilitated using Semipros 8.5 software.Furthermore, a secondary analysis, focusing on sensitivity, was conducted to provide additional insights into the robustness and potential variations in the results.
This multifaceted approach enhances the reliability and depth of the experimentation and subsequent analysis.
•  stillage is diverted towards SCP production alongside ethanol manufacturing, resulting in the generation of both ethanol and protein-rich fungal biomass.
It is crucial to clarify that in this integrated process, the traditional production of DDGS is replaced by the production of SCP.Therefore, rather than solely producing DDGS from whole stillage, a portion of the stillage is utilized for SCP production, leading to the generation of valuable proteinrich biomass in addition to ethanol.This integration not only diversifies the product portfolio but also potentially enhances the overall economic viability of the process, as indicated by the rise in net present value (NPV) and profit margin observed in that study 177 .Such an integration not only enhanced technical efficiency but also demonstrated economic advantages, with increased NPV and profit margins.Another study explored the technoeconomic viability of SCP production from grass silage using different techniques for biomass pretreatment, e.g.enzymatic hydrolysis, steam explosion, and alkaline pretreatment.The analysis considered CAPEX, OPEX (operational expenses), and protein quality.The study emphasized the needs such as enzyme costs, silage protein, and protein quality for commercial success 178 .These examples show the possible economic benefits of integrating SCP production into existing industrial processes, emphasizing the importance of considering both technical and economic aspects for sustainable and economically viable outcomes.
Commercial endeavors in the field of SCP have gained momentum as industries recognize the potential of this alternative protein source 179 .SCP derived from microbial biomass offers a sustainable and efficient solution to address the increasing global demand for protein 180 .
The fundamental condition of such use of SCP for feed and food is the demonstration of the absence of toxic compounds originating from substrates, biosynthesized by microorganisms, or formed during technological processes 181 .Different microorganisms may have varying capacities to metabolize substrates and produce specific compounds, influencing the safety and quality of the resulting SCP products.Furthermore, SCP preparations may vary in their potential to cause gastrointestinal disorders or allergic reactions, depending on the microorganism used and the composition of the final product 182 .Understanding the composition of microbiological cells, including their content of proteins, amino acids, lipids, vitamins, and minerals, is crucial for assessing the nutritional value and safety of SCP products 183 .
Currently, several multinational corporations and startups are involved and are starting new SCP-based industries, because human consumptive habits have changed and demand meets availability 184 .Global hotspots in the alternative protein space are Singapore and Israel, with commercial activities in Asia, the Americas, and Europe.Microorganisms like bacteria and fungi are commonly utilized by these industries for SCP production, with advancements in genetic engineering enabling the customization of strains to enhance productivity and nutritional content.The market players are diverse, and they follow several types of proteins for human and animal nutrition from different microorganisms, with significant variations in nutritional profiles and production methods 185 .Additionally, household industries are developed in villages with the support of the government and women's empowerment groups (V-HGsvillage self-help groups) for the production of mushrooms and yogurt, showcasing the versatility of microorganisms in SCP production 186 .
The expenses linked to single-cell protein depend on its designated use (whether for food or feed), the chosen quality of SCP, and competition from other protein products in terms of pricing 24 .SCP must be competitively priced compared to commercial animal and plant proteins, considering factors like price, nutritional value, and adherence to safety standards for both human and animal consumption [187][188][189][190][191] .In general, integrating microorganism/source-specific knowledge and features into the discussion of SCP production provides a more comprehensive understanding of the diverse applications and considerations associated with this promising protein source.
Precision fermentation has also emerged as a key area of innovation, to produce specific proteins with tailored functionalities 192 .These precision fermentation technologies hold promise for a wide range of applications spanning from food and pharmaceuticals to materials science, offering unprecedented opportunities for protein customization and optimization 193 .
Furthermore, the biofuel industry has seen significant advancements facilitated by genetically engineered yeasts 194,195 capable of fermenting cellulose to produce second and higher-generation biofuels.This innovative approach not only offers a sustainable alternative to traditional bioethanol production but also contributes to mitigating the environmental impact of fossil fuels, marking a critical step towards a more sustainable energy future 196 .However, despite the promising developments in single-cell protein production, scaling up from lab and pilot studies to industrial levels presents formidable challenges 197 .Optimizing fermentation conditions, ensuring product purity and consistency, and addressing economic feasibility are among the key considerations in this endeavor.Moreover, the transition to commercial-scale production requires substantial investments in infrastructure, technology, and process optimization 198 .Addressing these challenges is essential to realizing the full potential of single-cell protein production and harnessing its benefits on a global scale 199 .
Single-cell proteins are growing as a revolutionary solution in the quest for sustainable protein sources, finding applications in both animal feed and direct human consumption 200 .The regulatory framework governing the production and utilization of SCP is pivotal to ensuring the safety, quality, and adherence to standards within the dynamic landscape of protein production 201 .
In the realm of animal feed, SCP is subjected to a comprehensive set of regulations.These encompass strict guidelines for composition and nutritional standards, including the requisite levels of essential nutrients and amino acids essential for animal health 202 .Compliance with these standards is crucial to ensuring that SCP-based feed formulations are nutritionally adequate and contribute positively to the well-being of livestock 203 .Safety considerations take precedence in feed regulations, with established limits on contaminants such as heavy metals, mycotoxins, and undesirable microorganisms 204 .Robust monitoring and testing protocols are mandated to consistently meet these safety standards, ensuring that SCP does not pose risks to either the animals consuming them or the consumers of animal products 205 .
Clear and accurate labeling practices are integral to feed regulations, informing consumers about the presence of SCP in animal products 206 .Additionally, traceability measures are enforced to identify and address potential issues promptly, facilitating recall procedures if necessary.These measures collectively contribute to the responsible integration of SCPs into animal feed, balancing innovation with safety and transparency 207 .Transitioning to the realm of direct human consumption, SCP encounters a distinct set of food regulations.Often categorized as novel foods, SCP undergoes rigorous regulatory approval processes involving comprehensive assessments of safety, nutritional value, and potential allergenicity 208 .To assess allergenicity, various experiments are typically conducted.These may include in vitro assays using serum from individuals with known allergies to assess immunoglobulin E (IgE) binding potential.Additionally, animal models such as mice or rats may be employed to evaluate immune responses upon exposure to the novel food.Human clinical trials are also crucial, involving controlled consumption studies with allergic individuals to monitor adverse reactions.Furthermore, bioinformatics analyses may be utilized to compare the amino acid sequences of proteins in the novel food to known allergens, providing insights into potential cross-reactivity 209 .It is important to consider regulatory frameworks such as EFSA's Qualified Presumption of Safety (QPS).This framework evaluates the safety of microorganisms intentionally added to the food chain, including SCP.Criteria for QPS include factors such as lack of pathogenicity and absence of transferable antimicrobial resistance 210 .Understanding and adhering to such criteria are essential steps in ensuring the safety of SCP-containing foods for human consumption.The goal is to ensure that SCP introduced into the human food chain is not only safe but also nutritionally beneficial 211 .Allergen declarations become paramount in SCP-containing foods, ensuring that individuals with allergies are well-informed and protected.This aligns with broader food safety standards, dictating stringent processing and hygiene practices to prevent contamination and guarantee the microbiological safety of the final food product 212 .Accurate and transparent labeling is a cornerstone of food regulations for SCP, providing consumers with information about SCP content, nutritional benefits, and allergen presence.This fosters informed consumer choices and contributes to the responsible marketing and consumption of SCP-based food products 213 .
The feed and food legislation for SCP plays a pivotal role in shaping the trajectory of these innovative protein sources 214 .Compliance with these regulations is not just a legal requirement but a cornerstone of responsible innovation, ensuring that SCPs contribute positively to the global protein supply chain in a safe, sustainable, and transparent manner 215 .As SCPs continue to evolve and gain prominence, ongoing regulatory scrutiny remains essential to address challenges and advancements in the field.In 2022, the company Solar Foods obtained Novel Food approval in Singapore, making it a pioneer in the modern SCP feed industry 216 .Several decades ago, before the rise of cheap imported soy, there was an SCP industry, organized under UNICELPE, the unicellular protein producers 217 .
The need for novel foods (NF) approval for SCP varies depending on their intended use and the regulatory requirements of the jurisdiction where they are marketed 218 .While SCPs intended for direct human consumption typically require NF approval due to their novel composition, production methods, or source materials, SCPs used exclusively in animal feed or industrial applications do not necessitate NF approval 219 .The approval process often involves a comprehensive safety assessment, including evaluations of allergenicity, toxicity, nutritional composition, and the presence of contaminants 220 .Companies and researchers developing SCP must navigate the regulatory landscape, providing robust scientific evidence to support safety and efficacy while complying with applicable regulations 143 .Understanding these regulatory nuances is essential for ensuring consumer safety, and regulatory compliance, and fostering innovation in sustainable protein production 134 .While novel food approval is required for many SCP when intended for direct human consumption, it may not be necessary for SCP used exclusively in animal feed or industrial applications 153 .The need for NF approval depends on the specific regulatory requirements of the jurisdiction where the SCPs are intended to be marketed 160 .
Besides novel food regulations, SCP are subject to various other regulations depending on their intended use 162 .For SCP intended for animal feed, regulations governing feed safety, composition, labeling, and contaminants apply 169 .For SCP intended for human consumption, additional regulations related to food safety, nutritional labeling, allergen declaration, and food additives may apply 182 .Legislation for traditional foods may apply to SCP depending on their composition, production methods, and history of human consumption.Single-cell proteins that meet the criteria for traditional foods may benefit from certain exemptions or streamlined regulatory pathways 185 .However, SCP are often considered novel foods due to their innovative production methods or novel sources, which may subject them to additional regulatory requirements 190 .There may be regulatory gaps in the oversight of SCP, particularly in emerging areas such as cellular agriculture and biotechnology 192 .These gaps may arise due to the rapid pace of technological advancements outpacing regulatory frameworks, differences in regulatory approaches between jurisdictions, and the complexity of assessing novel food safety and environmental impact 125 .Addressing these gaps requires continuous dialog between regulators, industry stakeholders, and scientific experts to develop adaptive and robust regulatory frameworks that ensure the safety, sustainability, and ethical use of SCP 186 .
The safety of SCP is ensured through a multifaceted approach encompassing stringent regulatory frameworks, comprehensive safety assessments, and robust monitoring and testing protocols 131 .In the realm of animal feed, SCP is subjected to strict regulations governing composition, nutritional standards, and safety considerations 163 .Compliance with these regulations is crucial to ensure that SCP-based feed formulations meet nutrition requirements and do not pose risks to the health of livestock or consumers of animal products 174 .Safety measures include limits on contaminants such as heavy metals, mycotoxins, and undesirable microorganisms, with monitoring and testing protocols mandated to consistently meet these safety standards 186 .Clear and accurate labeling practices inform consumers about the presence of SCP in animal products, while traceability measures facilitate prompt identification and recall procedures if necessary 97 .
Transitioning to direct human consumption, SCP undergoes rigorous regulatory approval processes, often categorized as novel foods 152 .Comprehensive safety assessments evaluate allergenicity, toxicity, nutritional value, and potential risks, including bioinformatics analyses to compare protein sequences with known allergens 196 .Regulatory frameworks such as EFSA's qualified presumption of safety (QPS) evaluate the safety of microorganisms intentionally added to the food chain, including SCP, based on factors such as lack of pathogenicity and absence of transferable antimicrobial resistance 207 .Adherence to such criteria is essential in ensuring the safety of SCP-containing foods for human consumption 88 .
Furthermore, broader food safety standards dictate stringent processing and hygiene practices to prevent contamination and guarantee the microbiological safety of the final food product 54 .Accurate and transparent labeling is integral, providing consumers with information about SCP content, nutritional benefits, and allergen presence, fostering informed consumer choices, and responsible marketing and consumption of SCPbased food products 56 .Overall, compliance with regulatory requirements, adherence to safety standards, and ongoing monitoring and scrutiny contribute to ensuring the safety of SCP throughout their production and utilization 154 .
In addition to its positive attributes, SCP presents limitations for both human and animal consumption.Notably, the heightened concentration of nucleic acids in SCP, surpassing that of traditional protein sources, poses challenges 44 .This elevated nucleic acid (NA) content can lead to increased serum uric acid levels and the potential formation of kidney stones, as well as contribute to the development of gout, emphasizing the need for caution in direct SCP utilization 206 , or NA removal Increased serum uric acid levels resulting from the elevated nucleic acid content in SCP can also contribute to a condition called hyperuricemia 107 .Hyperuricemia is a medical term for high levels of uric acid in the blood, which can potentially lead to other health issues such as the deposition of urate crystals in tissues (tophi), joint inflammation (arthritis), and kidney damage over time 113 .Therefore, hyperuricemia presents another health concern associated with the consumption of SCP due to its nucleic acid content.Despite the majority of nitrogen in SCP being in the form of amino acids, the presence of nucleic acids, particularly prevalent in rapidly growing microorganisms, raises concerns and hinders the seamless integration of SCP as a widespread protein source 123 , necessitating NA removal Regarding safety requirements for SCP production, it is important to consider regulatory frameworks such as EFSA's QPS.This framework evaluates the safety of microorganisms intentionally added to the food chain, including those used in SCP production.Criteria for QPS include factors such as lack of pathogenicity, absence of transferable antimicrobial resistance, and generally recognized as safe (GRAS) status of the host organisms.To address concerns regarding SCP consumption, researchers can conduct analytical assays to quantify nucleic acid content, followed by animal feeding trials and clinical studies to assess SCP's impact on kidney function, serum uric acid levels, and kidney stone formation.Additionally, in vitro experiments and epidemiological studies can provide further insights into SCP's effects on cellular mechanisms and population health, aiding in understanding its overall safety profile and potential risks 56 .Understanding and adhering to such criteria are essential steps in ensuring the safety of SCP products for both human and animal consumption.Singlecell protein also contains a non-digestible cell wall, rendering it unsuitable for simple-stomach (monogastric) animals and birds 76 .Consumption of unprocessed SCP with active microbes may result in skin and gastrointestinal infections, causing nausea and vomiting.Filamentous fungi, with higher growth rates than yeasts, present contamination risks, while bacteria, with increased RNA content and contamination risks, pose limitations 34 .Certain microorganisms utilized in SCP production can produce toxic substances, including mycotoxins and cyanotoxins 106 .Consequently, carefully selecting microorganisms becomes crucial in SCP production to mitigate potential risks 134 .The presence of these substances, both in the substrate and the final product, raises concerns about indigestion and the potential formation of carcinogenic compounds, particularly during processing-related mutations 23 .Therefore, a cautious approach is essential in selecting microorganisms and processing techniques to guarantee the safety and integrity of SCP for consumption.Algae, while toxin-free, have a slow growth rate 76 .Addressing these limitations in SCP production involves optimizing fermentation protocols, selecting suitable microorganisms and substrates, and applying physical and chemical treatments 11 .
Recently, the adoption of LCCA has surged in economic analysis, aligning with the established standards of LCA 44 .This approach expands the horizon of cost analysis by incorporating externalities, particularly greenhouse gas emissions.Social life cycle assessment (SLCA) assumes a pivotal role in sustainability decisions, scrutinizing the social burdens or benefits entwined with a system.Social life cycle assessment delves into diverse social indicators, offering a qualitative portrayal of the social implications on workers, communities, consumers, and various stakeholders along the value chain 22 .The nascent concept of "environmental nutrition," integrated into food and nutrition assessments, seeks to harmonize sustainability by simultaneously considering environmental and nutritional benefits 56 .Life cycle sustainability assessment provides a holistic view by amalgamating all sustainability metrics and applying decision analysis for optimal solutions.Despite the evident significance of these concepts in sustainability assessment, their direct application in SCP production systems remains limited, presenting an avenue for refining the SCP process and product design and facilitating sustainable decision-making across different levels.
Sustainable food security implies physical and economic access to food for all.This requires greater efforts directed at food production and distribution as well as improving the living standards of the people.In the West, interest in healthy diets and novelty food is helping to drive a new interest in SCP.The forms in which SCP may be consumed are continuing to evolve.The chemical composition of any SCP product must be characterized clearly in terms of percentage protein, type of amino acids, nucleic acid, lipids, fats, toxins, and vitamins.A microbiological description indicating species, strains, and percentage of contaminants, if any, should be indicated.Final products for human consumption must be made to undergo rigorous testing during the pre-marketing stage and obtain novel food approval.Possible toxic compounds must be assayed for and removed.It is of primary importance that SCP products are safe to eat and also inexpensive to be popular among the masses.Further, genetically improved, high-yielding, and non-pathogenic microbes can be grown for SCP production.We should therefore look forward to extensive use of value-added SCP products in the new millennium and the eradication of chronic malnutrition globally by bridging the gap between demand and supply with SCP, which can also be a solution in a global agricultural crisis 221,222 , and having a broad range of food products that incorporate SCP should encourage further expansion of the market.

Fig. 1 |
Fig. 1 | Bibliometric analysis for the main keywords used in this study.The size of nodes shows the frequency of recurrence of the terms, and the connections between the different nodes show their simultaneous occurrence in a piece of literature.

75 )EligibilityFig. 2 |
Fig.2| Preferred reporting items for systematic reviews and meta-analyses (PRISMA) flow diagram of this study.This includes studies identified through other means, such as manual searches of reference lists, conference proceedings, or expert consultations.

Fig. 4 |
Fig.4| Micro-scale processes in solid-state fermentation (SSF) with Aspergillus oryzae: oxygen transfer dynamics.This scheme illustrates the micro-scale processes occurring during SSF involving fungi.The top images showcase the fermentation of wheat bran with Aspergillus oryzae.In SSF systems, the transfer of oxygen is limited by the liquid film on the substrate surface, with the primary source of oxygen coming from the gas phase14 .

Fig. 5 |
Fig.5| Classification of traditional fermented foods and beverages based on substrate and nutritional content.This classification scheme organizes conventional fermented foods and drinks according to their primary substrates and nutritional content99 .

Fig. 6 |
Fig. 6 | Simplified flow diagram for biomass-to-ethanol process utilizing enzymatic hydrolysis and simultaneous saccharification and confirmation.This diagram presents a streamlined overview of the biomass-to-ethanol process, highlighting enzymatic hydrolysis and the combined steps of saccharification and fermentation 114 .

Fig.
Fig.8| Guidelines for expressing case proteins and sgRNA in the CRISPR/Cas system.Guidelines for expressing Cas proteins and sgRNA within the CRISPR/Cas system.In the CRISPR/Cas9 system (A), the Cas9-sgRNA complex initiates a double-strand break (DSB) close to a protospacer adjacent motif (PAM), typically "NGG" for Cas9 from S. pyogenes.To efficiently target the nucleus in eukaryotes (B), it is recommended to fuse Cas9 with a nuclear localization sequence (NLS).The CRISPR/Cas12a (Cpf1) system (C) induces a DSB akin to Cas9 but relies on a

Table 1 |
8verage compositions of the main groups of microorganisms (% dry weight)8

Table 2 |
Summarizes the use of algae in different global regions npj Science of Food | (2024) 8:66

Table 5
furnishes a thorough summary of findings derived from various LCA studies on SCP, encapsulating their scope, methodologies, outcomes, gaps, and recommendations.Significantly, the table underscores the noteworthy environmental advantages associated with SCP, particularly in terms of global warming and land use.This underscores SCP's potential as

Table 5 |
Summary of SCP-LCA studies